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Title SUBSTRATES, STRUCTURES, AND FUNCTIONS OF THE CHLOROPLAST CLP PROTEASE SYSTEM IN ARABIDOPSIS THALIANA
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Publication Date
Date Accessioned
Degree PhD
Discipline/Department Plant Biology
Degree Level doctoral
University/Publisher Cornell University
Abstract The caseinolytic proteolytic machinery (CLP) is an essential and abundant protease of the chloroplast protease network. It is composed of multiple components (a proteolytic core CLPP/R/T, chaperones CLPC1/C2/D, and adaptors CLPS1/F). Mostly based on functional and structural information from bacterial Clp systems, it is postulated that these chloroplast CLP chaperones are aided by the CLP adaptors to select and deliver substrates to the proteolytic chamber (protease core) for degradation. The chloroplast CLPPRT proteolytic core is different and far more complex than the bacterial or mitochondria Clp core. The chloroplast CLP core is a hetero-oligomeric tetradecamer that is associated with additional accessory proteins unique to higher plants. Furthermore, the chloroplast CLPP and CLPR subunits have C-terminal extensions with unknown functions. It is unclear why chloroplast CLP core shows such high complexity and how these different CLP subunits contribute to the proteolytic system. Finally, relatively few chloroplast CLP substrates have been identified. To better understand the chloroplast CLP protease system, I applied an in vivo trapping approach for substrate identification and crosslinking (XL) mass spectrometry (MS) for investigation of the proximity and possible protein-protein interactions between these CLP components. Functional complementation showed that CLPP5 is crucial for CLP catalysis, whereas CLPP3 plays an essential role in CLP structure but its catalytic activity is dispensable. However, in vivo trapping using CLPPRT complexes with a reduced number of catalytic triads through the presence of one or more catalytically inactivated CLPP3/5 subunits did not identify proteins trapped in these CLPPRT complexes. This suggests that reduced proteolytic capacity within CLP cores does not result in a bottleneck for protein degradation in vivo. XL-MS of affinity-purified CLP core complexes or affinity purified CLPC-TRAP complexes identified several putative domains and motifs involved in the CLP protein-protein interactions. The newly established workflow of in vitro DSSO crosslinking using plant proteins paves the way for a more detailed exploration of the 3D structure and possible regulation of the chloroplast CLP machinery.
Subjects/Keywords CLP; subtrate; trapping; Proteolysis; Biochemistry; Botany; protease; Plant sciences
Contributors Van Wijk, Klaas (chair); Hanson, Maureen R. (committee member); Qian, Shu-Bing (committee member)
Language en
Country of Publication us
Record ID handle:1813/67299
Repository cornell
Date Retrieved
Date Indexed 2020-09-09
Grantor Cornell University
Issued Date 2019-05-30 00:00:00

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